Hsp40 interacts directly with the native state of the yeast prion protein Ure2 and inhibits formation of amyloid-like fibrils
Hsp40 interacts directly with the native state of the yeast prion protein Ure2 and inhibits formation of amyloid-like fibrils
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Hsp40 直接与酵母朊病毒蛋白 Ure2 的天然状态相互作用并抑制淀粉样蛋白样原纤维的形成
DOI：
10.1074/jbc.m606856200
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发表时间：
2007-04-20
影响因子：
4.8
通讯作者：
Perrett, Sarah
中科院分区：
文献类型：
--
作者：
Lian, Hui-Yong;Zhang, Hong;Perrett, Sarah
Ure 2是酿酒酵母中[URE 3]朊病毒表型的蛋白质决定簇，由柔性N-末端朊病毒决定结构域和球状C-末端谷胱甘肽转移酶样结构域组成。已发现I型Hsp 40成员Ydj 1在酵母细胞中的过表达导致[URE 3]的丢失。然而，Ydj 1治疗朊病毒的机制仍不清楚。在这里，我们测试了Hsp 40成员Ydj 1，Sis 1和Apj 1以及Hsp 70共伴侣Cpr 7，Cns 1，Sti 1和Fes 1在体内过表达的效果，发现只有Ydj 1对[URE 3]显示出强烈的治疗效果。我们还研究了Ydj 1与Ure 2在体外的相互作用。我们发现，Ydj 1是能够抑制淀粉样纤维的形成尿素2通过延迟原纤维形成的过程中，监测硫磺素T结合和原子力显微镜成像。使用牛血清白蛋白、Sis 1或人Hsp 40同源物Hdj 1或Hdj 2的对照没有显示出显著的抑制作用。Ydj 1是唯一有效的，当加入在原纤维形成的滞后期，这表明它与尿素2在原纤维形成的早期阶段相互作用，并延迟成核过程。使用表面等离子体共振和尺寸排阻色谱，我们证明了Ydj 1和野生型和N-末端截短的Ure 2之间的直接相互作用。相反，Hdj 2，它不抑制原纤维的形成，没有显示这种相互作用。结果表明，Ydj 1抑制Ure 2纤维的形成，通过结合到天然状态的Ure 2，从而延迟寡聚化的发生。
Ure2 is the protein determinant of the [ URE3] prion phenotype in Saccharomyces cerevisiae and consists of a flexible N-terminal prion-determining domain and a globular C-terminal glutathione transferase-like domain. Overexpression of the type I Hsp40 member Ydj1 in yeast cells has been found to result in the loss of [ URE3]. However, the mechanism of prion curing by Ydj1 remains unclear. Here we tested the effect of overexpression of Hsp40 members Ydj1, Sis1, and Apj1 and also Hsp70 co-chaperones Cpr7, Cns1, Sti1, and Fes1 in vivo and found that only Ydj1 showed a strong curing effect on [ URE3]. We also investigated the interaction of Ydj1 with Ure2 in vitro. We found that Ydj1 was able to suppress formation of amyloid-like fibrils of Ure2 by delaying the process of fibril formation, as monitored by thioflavin T binding and atomic force microscopy imaging. Controls using bovine serum albumin, Sis1, or the human Hsp40 homologues Hdj1 or Hdj2 showed no significant inhibitory effect. Ydj1 was only effective when added during the lag phase of fibril formation, suggesting that it interacts with Ure2 at an early stage in fibril formation and delays the nucleation process. Using surface plasmon resonance and size exclusion chromatography, we demonstrated a direct interaction between Ydj1 and both wild type and N-terminally truncated Ure2. In contrast, Hdj2, which did not suppress fibril formation, did not show this interaction. The results suggest that Ydj1 inhibits Ure2 fibril formation by binding to the native state of Ure2, thus delaying the onset of oligomerization.